Mechanical characterization data of polyacrylamide hydrogel formulations and 3D printed PLA for application in human head phantoms

To study human traumatic brain injury (TBI) mechanics, a realistic surrogate must be developed for testing in impact experiments. In this data brief, materials used to simulate brain tissue and skull are characterized for application in a full-scale human head phantom. Polyacrylamide hydrogels are implemented as tissue scaffolds and tissue mimics because they are bioinert and tunable. These properties make them ideal for use as brain tissue in studies that simulate head impacts. The objective is to modify hydrogel formulations to have minimal swelling and optical clarity while maintaining properties that mimic brain tissue, such as density, viscoelastic properties, and rheological properties. Secondly, polylactic acid (PLA) polymers are 3D printed to create biomimetic skulls to enclose the hydrogel brain tissue mimic or brain phantom. PLA samples are printed and tested to determine their mechanical strength with the intention of roughly matching human skull properties. Hydrogel data was obtained with an oscillatory rheometer, while PLA samples were tested using a mechanical tester with a 3-point bend setup. The present data brief highlights several hydrogel formulations and compares them to identify the benefits of each formula and reports mechanical values of 3D printed PLA samples with 100% grid infill patterns applied in a skull model.


a b s t r a c t
To study human traumatic brain injury (TBI) mechanics, a realistic surrogate must be developed for testing in impact experiments. In this data brief, materials used to simulate brain tissue and skull are characterized for application in a full-scale human head phantom. Polyacrylamide hydrogels are implemented as tissue scaffolds and tissue mimics because they are bioinert and tunable. These properties make them ideal for use as brain tissue in studies that simulate head impacts. The objective is to modify hydrogel formulations to have minimal swelling and optical clarity while maintaining properties that mimic brain tissue, such as density, viscoelastic properties, and rheological properties. Secondly, polylactic acid (PLA) polymers are 3D printed to create biomimetic skulls to enclose the hydrogel brain tissue mimic or brain phantom. PLA samples are printed and tested to determine their mechanical strength with the intention of roughly matching human skull properties. Hydrogel data was obtained with an oscillatory rheometer, while PLA sam-ples were tested using a mechanical tester with a 3-point bend setup. The present data brief highlights several hydrogel formulations and compares them to identify the benefits of each formula and reports mechanical values of 3D printed PLA samples with 100% grid infill patterns applied in a skull model.

Value of the Data
• The data presented characterizes equilibrated PAA hydrogel formulations tuned to match ex vivo brain tissue and in vivo human brain magnetic resonance elastography data. These formulations may be utilized in other tissue modeling applications. • The data characterizes the flexural strength of a 3D printed PLA skull model with 100 % grid infill. • Variations of hydrogel formulations exhibited in the data may be utilized as tissue scaffolds for cell types that are responsive to substrate stiffness for differentiation. • This brief provides a foundation to utilize these hydrogels as a tissue scaffold and to further characterize these PAA hydrogels at the microscale. • The brief addresses issues in the literature regarding the high variation of rheological data in hydrogels and the limited data on hydrogels with high polymer content.

Objective
This data brief supports the publication titled 'Characterization of Material Properties and Deformation in the ANGUS Phantom During Mild Head Impacts' by providing the foundational mechanical characterization data necessary to fabricate a head phantom with specified properties to analyze simulated mild head impacts [1] . This data brief characterizes two cranial components, brain tissue and skull. PAA hydrogels are used as a biomaterial because they are bioinert and tunable [2] . In the literature, hydrogel properties and formulations are highly variable making it difficult to reproduce. Additionally, limited data is available on hydrogels with high polymer content. Variations in monomer and crosslinker content, temperature, and linear acrylamide chains provide different properties that can meet desired specifications, such as swelling, optical clarity and rheological properties. Monomer to crosslinker formulations of 60-1 provide optical clarity but swell significantly, while 15-1 formulations have minimal swelling but are turbid. An ideal formulation will minimally swell and remain optically clear for high-speed imaging. These formulations, for white and gray matter, achieve a storage modulus of 20 0 0 or 1400 Pa, a loss modulus of 400 or 300 Pa, respectively, and a tan δ of 0.2 [1 , 3] . Hydrogels with increased linear acrylamide chains were formulated in an effort to obtain an increased viscous component thereby increasing the tan δ, the ratio of viscous to elastic forces [4] . An important property of the skull is its ability to deform before it fails, the flexural modulus is analyzed in 3-point bend tests on PLA samples with 100% grid infill to mimic skull behavior.

Data Description
Two categories are reported, PAA characterization to represent brain tissue and 3D printed PLA parts to represent the skull in the human head surrogate. The rheology, optical, and swelling data are all contained in the ' Hydrogel Rheology, Optical Values, and Swelling' repository [5] . Figures 1-3 display rheological data, the file names in the repository describe which formulation was used by specifying the acrylamide weight percent and crosslinker ratio. In this brief, weight percent is written as wt. % and is defined as the percent weight per volume. The file name also specifies samples that were heated or used linear acrylamide chains [5] . UV spectroscopy and hydrogel swelling data are also included. The UV spectroscopy excel file has each sample in an individual sheet and includes the wavelength and absorbance values, the file name, and the time and date on which the test was conducted [5] . Lastly, the swelling data is included in an excel file that has the time in hours which is highlighted in green. The average and standard deviation values of the three dimensions and the volume are calculated for each time point in centimeters [5] . Three Python scripts are also included in the repository, two rheology scripts, and one swelling script. The two rheology scripts and MATLAB script were hard queried for certain data files, all of which are included in the repository. Both rheology scripts average the desired data, and all scripts used the matplotlib library for visualization.
The ' 3-Point Bend Testing for 100% Grid Infill PLA Samples' data repository includes three files, two excel files which contain six sheets. The first five sheets are the individual samples that are collected including their load, travel, time, and calculated values for the load versus displacement and flexural modulus [6] . The sixth sheet contains the average and standard deviation for all samples. The associated MATLAB script evaluates one excel file at a time and using known values of depth, support span, and width calculates strain and stress based on calculations obtained from ASTM D790 [6 , 7] . % monomer content. Hydrogel raw data sets, available in the data repository associated with this article, show that most formulations had n = 10 with the exceptions of 5 wt. % 15-1 (n = 8) and 10 wt. % 15-1 (n = 9) [5] . The excluded samples in those two formulations were due to hyperelastic behavior caused by a stress setting beyond the yield stress of the sample. Fig. 1a shows the average storage modulus of 60-1 versus 15-1 formulations at different monomer (wt. %) content where 15-1 hydrogels with similar monomer content provide a higher storage modulus. A similar comparison is apparent in the loss modulus ( Fig. 1b ). The tan δ for 60-1 and 15-1 formulations is in Fig. 1c . Both formulations obtain a range of rheological properties desired for tissue scaffold and brain mimic applications. Based on Magnetic Resonance Elastography of healthy volunteers and indentation measurements of the bovine brain it was determined that an ideal hydrogel formulation to mimic storage and loss modulus of brain tissue would be 20 0 0 Pa and 400 Pa for white matter and 1400 Pa and 300 Pa for gray matter, respectively, that would result in a tan δ of 0.2 [1 , 3] . Fig. 1 was created with the Python script, ' Rheology -Hydrogel Monomer and Crosslinker Comparison' which was used to analyze six files (Rheology_10_wt_15_1, Rheology_10_wt_60_1, Rheology_12_wt_15_1, Rheology_5_wt_15_1, Rhe-ology_7_wt_15_1, Rheology_7_wt_60_1). All these files are included in the data repository titled ' Hydrogel Rheology, Optical Values, and Swelling' [5] .   to DI water [4] . These chains are created by pre-polymerizing the monomer into longer chains before crosslinking (see Linear Acrylamide Hydrogel Formulations in Methods). Raw data sets corresponding to Fig. 2 are available in the data repository associated with this article [5] . Fig. 2 compares 60-1 and 15-1 monomer to crosslinker ratios with 10 wt. % monomer content and 15 wt. % linear acrylamide chains hydrogel formulations. Figs. 2a and 2b demonstrate that higher crosslinker formulations (15-1) provide higher storage and loss values compared to lower crosslinker formulations (60-1) as expected. Fig. 2c displays that the tan δ of linear acrylamide hydrogels is lower than that of 60-1 and 15-1 formulations without linear acrylamide chains. Fig. 2 was created with the Python script, 'Rheology_LinearAnalysis' which analyzes two  files (Rheology_10_wt_15_1_15linear, Rheology_10_wt_60_1_15linear) . Both files are included in the data repository titled 'Hydrogel Rheology, Optical Values, and Swelling' [5] . Fig. 3 shows the average a) storage, b) loss, and c) tan δ values obtained in a dynamic frequency sweep (n = 9). Raw data sets corresponding to Fig. 3 are available in the data repository associated with this article [5] . Fig. 3 formulations are comparing heated hydrogel formulations of 10 wt. % and 5 wt. % acrylamide and 15-1 crosslinker ratio. Polymerizing hydrogels at 60 °C dissociates the initiator (ammonium persulfate) into free radicals meaning bonds are broken which results in gels that are more optically clear and have lower storage and loss modulus values than non-heated formulations [8] . The tan δ value of 0.2 was near the desired value. Fig. 3 was developed with the Python script, 'Rheology -Hydrogel Monomer  and Crosslinker Comparison' which analyzes two files, 'Rheology_Heated_Hydrogels_10wt_15_1' and 'Rheology_Heated_Hydrogels_5wt_15_1' both of which are included in the data repository titled 'Hydrogel Rheology, Optical Values, and Swelling' [5] . Fig. 4 displays the difference in swelling between different crosslinker ratios, 15-1 provides the least amount of swelling while 60-1 has the most. There are minor deviations in measurement because of user error, average volume was used to monitor swelling and changes in dimensions were measured using digital calipers. 60-1, 45-1, and 15-1 formulations had n = 6 and the 30-1 formulation had n = 4. Fig. 4 was visualized with a Python script titled 'Swelling_10wt'  which analyzed the file 'Swelling_Data' included in the data repository titled 'Hydrogel Rheology, Optical Values, and Swelling' [5] . Fig. 5 compares different hydrogel formulations' light absorbance with a path length of 1 cm from the file 'UV_Vis_Reading´included in the data repository 'Hydrogel Rheology, Optical Values, and Swelling' [6] . Higher crosslinker formulations (15-1) had more light absorbance than lower crosslinker formulations (60-1) because of the higher density of bonds in 15-1 formulations. The 10 wt. % 60-1 is hidden behind the 7 wt. % 60-1, this stems from the large absorbance axis limits to include the 15-1 data and also because of the similar clarity in 60-1 formulations at these monomer values. However, when 15-1 formulations were polymerized at 60 °C instead of  room temperature light absorbance was lower than 15-1 formulations and more comparable to 60-1 formulations because of the dissociation of the initiator into free radicals [8] . Fig. 6 shows the average flexural stress for PLA samples with a 100% grid infill (n = 5) used in the skull of brain phantoms, with the 'X' signifying the fracture point [1] . Literature values for the flexural modulus of human skull is reported at 11.73 GPa [9] . Samples were tested with the top roller approaching the sample with a displacement rate of 3.41 and 10 mm/min. The raw data associated with this graph is present in the data repository associated with this article. Flexural modulus was calculated in excel with E B = L 3 m 4 b d 3 where L = support span (meters), m = slope of displacement-load curve, b is the width of the sample (meters), and d is the thick-  [7] . The 10 mm/min samples had a flexural modulus of 2.86 GPa ± 0.151, and the 3.4 mm/min samples had a flexural modulus of 2.94 GPa ± 0.032 which are acceptable values for the current skull model. The MATLAB script 'Skull_10mm' was used to analyze the file for Fig. 6a , '100%Grid_PLA_3PB-3.4mm-min' and Fig. 6b , '100%Grid_PLA_3PB_10mm-min' [6] . Both of these files are available in the data repository titled '3-Point Bend Testing for 100% Grid Infill PLA Samples' [6] . Although the MATLAB script specifies 10 mm, it is a general script that can assess samples of any speed setting.

Hydrogel Formulations
Four hydrogel variables were tested, monomer to crosslinker ratios (60-1 and 15-1), 5 -12 wt. % monomer content, hydrogels with linear acrylamide chain content, and hydrogels polymerized at 60 °C. The formulations with varied monomer content and monomer to crosslinker ratio followed the same basic protocol, deionized (DI) water is degassed and stirred for 10 minutes. Then acrylamide, methylenebisacrylamide (MBA), and the initiator ammonium persulfate (APS) are all added independently and degassed with a vacuum pump, and stirred for 3, 10, and 2 minutes respectively. Tetramethylethylenediamine (TEMED), acting as a catalyst, is added to the gel precursor solution to induce polymerization at room temperature. As an example, for a 100 mL batch of 10 wt. % 15-1 hydrogel 10 grams of acrylamide, 0.667 grams of MBA, and 0.0867 grams of APS are used with 180 μL of TEMED [10] . Monomer content is based on weight per volume and MBA is calculated based on desired crosslinker ratio, which in this example is 15-1. The APS and TEMED values are calculated with the following equations based on values used by Wermer, A., et al. (2020) [10] .

Heated Hydrogel Formulations
Heated hydrogels were made with a similar protocol, DI water was heated, degassed, and stirred for 10 minutes. Acrylamide and MBA are added simultaneously and heated, degassed, and stirred for three minutes. After this period degassing and stirring is stopped and the temperature is brought to 60 °C, then APS and TEMED are added simultaneously and stirred for 10 seconds. During all of this, the designated molds are heated to 60 °C in a furnace. The gel-precursor solution is then added to the mold and placed back inside the furnace during polymerization.

Linear Acrylamide Hydrogel Formulations
Linear acrylamide hydrogels were prepared in two phases, the first is to prepare a linear acrylamide solution and the second is to polymerize the hydrogel. DI water is degassed and gently stirred for 10 minutes, then acrylamide is added based on the desired wt. % and degassed and gently stirred for 3 minutes. The solution is then heated to 37 °C then APS and TEMED are added to form linear chains. The linear chain solution is maintained at 37 °C and stirred for 2 hours. After that time, the solution is cooled to room temperature and stored in 4 °C until needed. For the second step, hydrogel polymerization, DI water is degassed and gently stirred for 10 minutes, and then acrylamide, MBA, linear acrylamide chains, and APS are added and degassed independently while being stirred for 3, 5, 3, and 2 minutes respectively. TEMED is added and the solution is allowed to gel for 24 hours.The acrylamide, MBA, APS, and TEMED values are based on the same values indicated in 2.1. The same amount of APS and TEMED are added for both the linear solution and the hydrogel polymerization. The equations for these values were indicated in '2.1 Hydrogel Formulations'.

Rheological Measurements
The SR5 was utilized to conduct oscillatory rheology on hydrogel samples. Swollen hydrogel discs with a 25 mm diameter and approximately 1 mm height were polymerized and swollen in an isotonic solution for 24 hours. The upper fixture of the instrument was lowered onto the sample to obtain an axial force between 0.01 -0.2 N. The stress setting for frequency sweeps was set under the yield stress of the sample, which was estimated by performing a stress sweep and finding the stress value where 1% strain was achieved. Hydrogels with a monomer to crosslinker ratio of 15-1 were tested without any sandpaper present to prevent slippage, whereas 60-1 hydrogels were tested with sandpaper. This is due to the different surface characteristics present on the 15-1 and 60-1 hydrogels. Data is reported to 10 Hz as inertial forces dominate the data beyond that.

Python Script
The rheological data was plotted with a simple Python script. Raw data is entered into excel and the Python script references the excel file to plot the data. This script calculates and plots the average and standard deviation of all samples in a batch.

Hydrogel Swelling
Hydrogel formulations were polymerized in cubic silicon molds approximately 3 cm in all dimensions. Hydrogel cubes were then removed from their molds and placed into an isotonic solution to swell. Cubes were measured with digital calipers and the average volume was calculated.

3D Printed Samples
PLA samples were 3D printed with a Creality CR-10S Pro V2. The settings of the printer were a 205 °C nozzle temperature and 40 mm/s print speed. The samples were made to have a geometry of 154 mm length, 12.7 mm width, and 8 mm thickness. In this geometry the wall count, top, and bottom layer count were all set at 2. These samples had an infill percentage of 100% with a grid pattern.

3-Point Bend Testing
The specimens were manufactured and tested via three-point bending according to the ASTM D790-10 standard which tests flexural properties of polymers [7] . The three-point bending tests were conducted using a Mark-10 ESM1500S at a crosshead speed of 3.4 mm/min and 10 mm/min. Each specimen was tested until break or ultimate flexural strength (UFS) was reached. The flexural modulus is obtained from the average of the five specimens. In excel the flexural modulus was calculated by calculating the slope of the linear portion of the flexural stress.

MATLAB Script
Methods and calculations were determined by ASTM standards [7] . The raw data from the 3-point bend testing apparatus is obtained and entered into excel. Load versus displacement curves is plotted from the output data from the mechanical tester.
In the MATLAB flexural stress of the samples are calculated by: